DNA sequences encoding for subunit CHLD of plant magnesium chelatases and determining the activity of plant magnesium chelatases

ABSTRACT

The present invention relates to a nucleic acid molecule which encodes a protein with the function of a plant Mg chelatase subunit CHLD or an active fragment thereof; a protein which has the function of a plant Mg chelatase subunit CHLD or an active fragment thereof, preferably a recombinant protein; a method of determining the interaction of plant Mg chelatase subunits, in which a host cell is transformed with a DNA sequence as claimed in one or more of claims 1 to 3 and at least with one DNA sequence encoding a further subunit of Mg chelatase in such a manner that the interaction of the Mg chelatase gene products leads to a directly or indirectly, qualitatively or quantitatively measurable signal, preferably by activating a marker gene, and transgenic plants, transgenic plant cells, transgenic plant organs, transgenic plant seeds, transgenic propagation material comprising an abovementioned nucleic acid molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from International Application No. PCT/EP98/02483, filed Apr. 27, 1998 and German Application No. 197 17 656.9, filed Apr. 25, 1997. Each of the foregoing applications, patents and publications and all documents cited or referenced therein (“application cited documents”) and all documents cited or referenced in this specification (“herein cited documents”) are hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The present invention relates to the subunit CHLD of plant magnesium chelatase (Mg chelatase), to DNA sequences encoding the subunit CHLD of plant Mg chelatase, to processes for preparing the subunit CHLD of plant Mg chelatase, to processes for determining the activity of plant Mg chelatase, and transgenic plants which have been transformed with the Mg chelatase DNA according to the invention.

BACKGROUND OF THE INVENTION

Being photosystem cofactors, chlorophylls play a role in the conversion of light into chemical energy and are thus required for plant growth and survival.

Magnesium is incorporated into porphyrin during chlorophyll biosynthesis with the aid of a membrane—associated enzyme, namely Mg chelatase, which is composed of several subunits.

It has already been disclosed that bacterial Mg chelatase is composed of three subunits (D, H and I), whose corresponding gene sequences are termed bchD, bchH and bchI (Burke et al. (1993), J. Bacteriol. 175, 2414-2422; Coomber et al. (1990), Mol. Microbiol. 4, 977-989; Gibson et al. (1995), Proc. Natl. Acad. Sci. USA, 92, 1941-1844; Jensen et al. (1996), J. Biol. Chem. 271, 16662-16667).

TABLE I List of the genes of known Mg chelatase subunits Rhodobacter Rhodobacter Synechocys- Arabidopsis Antirrhinum capsulatus sphaeroides tis PCC 6803 thaliana majus Burke et al. Coomber et al. Jensen et al. Koncz et al. Hudson et al. bchD bchD chID bchH bchH chIH chiH olive bchl bchl chil ch42 3Dchl

As regards plant Mg chelatases, two subunits have been described to date which seem to correspond to the bacterial Mg chelatase subunits bchH and bchI (Koncz et al., (1990), EMBO J. 9, 1337-1346; Hudson et al., (1993), EMBO J. 12, 3711-3719; Eibson et al., (1996), Plant Physiol. 121, 61-71). It is not known as yet which other subunits participate in the structure of plant Mg chelatase. No enzyme activity was observed with the two known plant subunits CHLI and CHLH, neither alone nor together with the known bacterial subunits of type D (CHLD and BCHD).

Due to their key position in chlorophyll biosynthesis, plant Mg chelatase is a radically new starting point for developing a novel generation of herbicidal compounds with highly specific activity. In addition, the vitality and/or growth of phototrophic uni- and multicellular organisms, in particular bacteria, algae and plants, can be controlled to a high degree by influencing gene expression (suppression, overexpression) of the natural or modified (for example genetically engineered) expression products of Mg chelatase or else by specific Mg chelatase inhibitors.

The enzymatic activity of plant Mg chelatase was originally measured on intact chloroplasts (Castelfranco et al., (1979) Arch. Biochem. Biophys. 192, 592-598; Fuesler et al. (1982) Plant Physiol. 69,421-423). Since then, the activity was also determined on disrupted plastids (Walker et al. (1991) Proc. Natl. Acad. Sci. 88, 5789-5793) and subplastid membrane fractions (Lee, et al. (1992). Plant Physiol. 99,1134-1140).

Surprisingly, there has now been found a DNA which encodes a subunit of the enzyme plant Mg chelatase. The DNA will subsequently be termed chlD, and the amino acid sequence CHLD.

Moreover, it has been found that a subunit CHLD of plant Mg chelatase together with the subunits CHLI and CHLH is, surprisingly, suitable for reconstituting a funtionally intact, i.e. enzymatically active, plant Mg chelatase, so that the plant Mg chelatase subunit CHLD according to the invention provides novel test methods (in vivo and in vitro) for plant Mg chelatase activity.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D depict transgenic antisense and sense CHLD tobacco plants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore relates to a nucleic acid molecule which encodes a protein with the function of a plant Mg chelatase subunit CHLD, preferably a nucleic acid molecule as shown in SEQ ID NO: 1, a biologically active fragment thereof, and complementary or antisense sequences.

The invention relates to plant chlD sequences, preferably from dicotyledonous plants, especially preferably Solanaceae and, in particular, Nicotiana tabacum.

The present invention futhermore relates to a nucleic acid molecule which encodes a protein in accordance with SEQ ID NO: 2 with the function of a plant Mg chelatase subunit CHLD or a biologically active fragment thereof or it forms a complementary or antisense sequence to such a nucleic acid molecule.

A further object of the present invention is a nucleic acid molecule which is an oligonucleotide of at least 10 nucleotides in length and which hybridizes specifically with a nucleic acid molecule which encodes a protein with the function of a plant Mg chelatase subunit CHLD, preferably SEQ ID NO: 1, a fragment thereof or its complementary or antisense sequences.

The term “to hybridize specifically” is generally understood to refer to the characteristic of a single-stranded nucleic acid molecule to form, together with a complementary nucleic acid molecule, hydrogen bridges, base pairs and, if appropriate, a double strand.

Another object of the present invention is a nucleic acid molecule which encodes a peptide from the group consisting of SEQ ID NO: 3, SEQ ID NO: and SEQ ID NO: 7, and their complementary or antisense sequences.

The term “recombinant organism” is to be understood as meaning the cell of an organism which has been modified by altering its DNA in vitro or by integration of DNA, for example recombinant yeast, bacterial, algal, insect or plant cells.

Writing “chI” in small letters is conventionally used for the genes of Mg chelatase subunits D, I and H, while the capitalization “CHL” refers to the gene products, i.e. proteins.

A further object of the present invention is the use of the nucleic acid molecules according to the invention for amplifying, isolating or identifying a nucleic acid molecule encoding a protein with the function of a plant Mg chelatase subunit CHLD or a biologically active fragment thereof, in particular of chlD structural genes or chlD mRNA of other organisms, preferably of plant or microbial origin.

The invention furthermore also relates to the use of the nucleic acid molecules according to the invention for raising antibodies against their expression products.

Another object of the invention are non-naturally occurring chimeric genes comprising a promoter which is functionally fused to a DNA molecule according to the invention.

Another object of the invention is a vector, preferably a recombinant vector, comprising a nucleic acid molecule according to the invention comprising a chimeric gene comprising a promoter, which is functionally fused to a DNA molecule according to the invention.

Another object of the invention is a recombinant host cell which expresses a DNA molecule according to the invention, this host cell preferably being stably transformed with a recombinant vector comprising a chimeric gene comprising a promoter which is functionally fused to a nucleic acid molecule according to the invention or which is transformed by other transformation methods known to those skilled in the art and expresses a DNA molecule according to the invention.

The invention furthermore relates to the use of the nucleic acid molecules according to the invention for generating transgenic plants.

Another object of the invention are transgenic plants, transgenic plant cells, transgenic plant organs, transgenic plant seeds, transgenic propagation material comprising a nucleic acid molecule according to the invention.

Another object of the invention are methods of generating transgenic plants, transgenic plant cells, transgenic plant organs, transgenic plant seeds, transgenic propagation material by means of recombinant host cell, wherein this host cell is transformed with a DNA molecule according to the invention.

The invention also relates to propagation material of the plants according to the invention, for example fruits, seeds, tubers, root stocks, seedlings and cuttings.

Suitable excipient plants for a gene according to the invention are all agriculturally important monocotyledonous and dicotyledonous crop plants, preferably maize and other cereals such as, for example, wheat, rye, barley, panic grasses, oats, cassava and rice, and also cotton, tobacco, sugar beet, sugar cane, potatoes, oilseed rape, sunflowers, soya, or fruit and vegetable species.

To express the nucleic acid molecules according to the invention in plant cells, they are linked to regulatory DNA elements which ensure transcription in plant cells. These include, in particular, promoters. In general, any promoter which is active in plant cells is suitable for expression.

The promoter may be chosen in such a way that expression is constitutive or only takes place in a certain tissue, at a certain point in time of the plant's development or at a point in time determined by external factors. As regards the plant, the promoter can be homologous or heterologous. Examples of suitable promoters are the cauliflower mosaic virus 35S RNA promoter and the maize ubiquitin promoter for constitutive expression, the patatin gene promoter B35 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for sink-specific expression (for example potato tubers, beet, tomato fruit) or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451), all promoters which are constitutively active in plastids, for example the psbA cassette expression signal sequences (Staub & Maliga (1993) EMBO Journal 12: 601-606; Zoubenko et al. (1994) Nucleic Acids Research 22: 3819-3824) and the Prn promoter (Svab+Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917) or, for endosperm-specific expression, the wheat HMG promoter, the USP promoter, the phaseolin promoter, or promoters from maize zein genes. Furthermore, a termination sequence may be present which serves for correctly terminating transcription and for adding a poly-A tail to the transcript, which is assumed to have a function in stabilizing the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and are, in general, exchangeable as desired.

A large number of cloning vectors are available for preparing the introduction of foreign genes into higher plants, and these cloning vectors contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184. The desired sequence can be introduced into the vector at a suitable restriction cleavage site. The plasmid obtained is used for transforming E. coli cells. Transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. Analytical methods for characterizing the plasmid DNA obtained are, in general, restriction analyses, gel electrophoreses and other methods of biochemistry and molecular biology. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned in the same or other plasmids.

A large number of techniques is available for introducing DNA into a plant host cell. These techniques encompass the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, protoplast fusion, injection, DNA electroporation, the introduction of DNA by means of the biolistic method and the like.

When injecting and electroporating DNA into plant cells, the plasmid used need not meet any particular requirements. Simple plasmids such as, for example, PUC derivatives, may be used. If, however, whole plants are to be generated from such transformed cells, the presence of a selectable marker gene is required.

Depending on the method by which genes are introduced into the plant cell, other DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for transforming the plant cell, at least the right hand side limit, but frequently the right and left hand side limit, of the Ti and Ri plasmid T-DNA must be linked, as a flanking region, with the genes to be introduced.

If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into specific plasmids, viz. either into an intermediary vector or into a binary vector. The intermediary vectors can be integrated by homologous recombination into the Ti or Ri plasmid of the agrobacteria due to sequences which are homologous to sequences in the T-DNA. The Ti or Ri plasmid also contains the vir region which is required for transferring the T-DNA. Intermediary vectors cannot replicate in agrobacteria. The intermediary vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors are capable of replication both in E. coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker which is framed by the left and right T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187). The agrobacterium which acts as the host cell should contain a plasmid which carries a vir region. The vir region is required for transferring the T-DNA into the plant cell; additional T-DNA may be present. This transformed agrobacterium is used for transforming plant cells.

The use of T-DNA for transforming plant cells has been described extensively in EP 120 516; Hoekema, in: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 146 and An et al. EMBO J. 4 (1985), 277-287.

To transfer the DNA into the plant cell, plant explants may advantageously be cocultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants may be regenerated from the infected plant material (for example leaf sections, stem segments, roots, but also protoplasts or suspension-cultured plant cells) in a suitable medium which may contain antibiotics or biocides for selecting transformed cells. The resulting plants can then be tested for the presence of the DNA which has been introduced. Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf., for example, Willmitzer, L., 1993 Transgenic Plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim).

Alternative systems for the transformation of monocotyledonous plants are the transformation by means of the biolistic approach, the electrically or chemically induced uptake of DNA into protoplasts, the electroporation of partially permeabilized cells, the macroinjection of DNA into inflorescences, the microinjection of DNA into microspores and proembryos, the DNA uptake by germinating pollen, and the DNA uptake in embyros by swelling (review: Potrykus, Physiol. Plant (1990), 269-273).

While the transformation of dicotyledonous plants via Ti-plasmid vector systems with the aid of Agrobacterium tumefaciens is well established, later publications suggest that even monocotyledonous plants are indeed accessible to transformation by means of Agrobacterium-based vectors (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282; Bytebier et al., Proc. Natl. Acad. Sci. USA 84 (1987), 5345-5349; Raineri et al., Bio/Technology 8 (1990), 33-38; Gould et al., Plant Physiol. 95 (1991), 426-434; Mooney et al., Plant Cell Tiss. & Org. Cult. 25 (1991), 209-218; Li et al., Plant Biol. 20 (1992), 1037-1048).

Three of the abovementioned transformation systems were established in the past for a variety of cereals: the electroporation of tissue, the transformation of protoplasts, and the DNA transfer by particle bombardment of regenerable tissue and cells (Jähne et al., Euphytica 85 (1995), 35-44).

The transformation of wheat is described in various references (review: Maheshwari et al., Critical Reviews in Plant Science 14 (2) (1995), 149-178), cf. also Hess et al. (Plant Sci. 72 (1990), 233), Vasil et al. (Bio/Technology 10 (1992), 667-674), Weeks et al. (Plant Physiol. 102 (1993), 1077-1084), and Becker et al. (Plant J. 5 (2) (1994), 299-307).

Once the DNA introduced is integrated in the genome of the plant cell, it is, as a rule, stable and is retained in the progeny of the originally transformed cell. It normally contains a selection marker which mediates resistance to a biocide such as phosphinothricin or an antibiotic such as kanamycin, G 418, bleomycin or hygromycin to the transformed plant cells. The marker, which is chosen individually, should therefore allow the selection of transformed cells over cells which lack the DNA introduced.

The transformed cells grow within the plant in the customary manner (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be grown normally and hybridize with plants which have the same transformed genetic material, or other genetic material. The resulting hybrids have the corresponding phenotypic characteristics. Seeds may be obtained from the plant cells.

Two or more generations should be grown in order to ensure that the phenotypic trait is stably retained and inherited. Also, seeds should be harvested to ensure that the relevant phenotype, or other characteristics, have been retained.

Another object of the invention are, furthermore, methods of generating a nucleic acid molecule according to the invention, wherein a nucleic acid molecule according to the invention is generated by a method known per se for the generation of nucleic acids. A wide range of different methods of isolating and cloning gene sequences are available to those skilled in cDNA cloning.

In addition, the sequence informations according to the invention can also be utilized for a multiplicity of other methods known to those skilled in the art, such as, for example, screening expression libraries for isolating genes using antibodies, or else for raising antibodies (polyclonal or monoclonal).

The entire plant chlD cDNA, preferably chlD from Nicotiana tabacum as shown in SEQ ID NO: 1, subsequences from SEQ ID NO: 1 or oligonucleotides derived from SEQ ID NO: 1 may be employed as probes for such methods, if appropriate those which selectively hybridize with other CHLD-encoding sequences from a library of cloned gene fragments of a selected organism, for example phototrophic microorganisms or mono- or dicotyledonous plants.

Such techniques include, for example, also the screening of genomic or cDNA libraries, cloned into suitable cells or viruses, and the amplification by means of polymerase chain reaction (PCR) using oligonucleotide primers derived from SEQ ID NO. 1.

The isolated chlD (sub)sequences according to the present invention can furthermore be modified or extended by standard methods of genetic engineering so as to obtain desired properties. To obtain selective hybridization under in-vitro or in-vivo conditions, chlD-specific probes have a length of at least 10 nucleotides, preferably at least 17 nucleotides.

Hybridization probes according to the present invention can be used, for example, to amplify or analyze CHLD-encoding sequences (i.e. CHLD-encoding nucleic acid molecules) from a selected organism by means of the PCR method, which is known to those skilled in the art, and can furthermore be used for isolating CHLD-encoding sequences from other organisms or as a diagnostic for detecting CHLD-encoding sequences in other organisms, or to identify and isolate modified CHLD-encoding sequences of a specific phenotype, such as, for example, resistance to herbicides, altered chlorophyll content, altered fitness, altered yield and the like.

The chlD-specific hybridiration probes can also be used to map the location of a naturally occurring chlD gene in a plant genome, using standard methods. These methods include, inter alia, identification of DNA polymorphisms and the use of such polymorphisms for analyzing the segregation of chlD genes in relation to other markers of known location. Mapping chlD genes may be of particular interest in plant breeding.

chlD-specific hybridization probes or CHLD-encoding (sub)sequences may also be exploited to inhibit expression of a chlD gene in planta (antisense technology).

Using standard methods, it is also possible to modify CHLD-encoding (sub)sequences or to extend them by novel sequence elements and to introduce them into the genome of plants. Plant transformation can be transient or stable, using conventional methods. The integration of sequences derived from chlD cDNA into the plant genome can be exploited for altering the properties of the plant, so that, for example, more or less functionally active Mg chelatase or a variant of Mg chelatase with altered properties is formed in the transgenic plant, or else that the expression level of the chlD gene in the transgenic plant is reduced. For example, the amount of functional Mg chelatase can be increased by standard methods if, for example, the chlD gene, alone or together with genes of other Mg chelatase subunits, is cloned into the transgenic plant under the transcriptional control of regulatory elements such as promoters and enhancers. An increased CHLD activity can be used, for example, for raising the chlorophyll content, which may be linked to higher yields, or to raise the tolerance to herbicides which inhibit chlorophyll biosynthesis. Also, for example cloning altered sequences according to the invention into transgenic plants, may be linked with a higher tolerance to herbicides.

The nucleic acid molecules which encode chlD sequences or fragments of these sequences (molecules) and which are introduced into transgenic plants can, however, also be provided with regulatory elements such as, for example, promoters and enhancers, in such a way that this results in lowering the original Mg chelatase activity in the transgenic plant (for example cosuppression, formation of chlD antisense RNA). A reduced Mg chelatase activity can be exploited for generating chlorotic plants or plants with chlorotic tissue. A reduced chlorophyll content may be desired, for example, when plants or plant organs are subjected to industrial processing at a later point in time, or when breeding for a variety of crops or ornamentals, for example vegetables such as chicory.

The present invention furthermore relates to a protein which has the biological function of a plant Mg chelatase subunit CHLD, preferably according to SEQ ID NO: 2, or a biologically active fragment thereof.

Another object of the invention is the use of a protein with the biological function of a plant Mg chelatase subunit CHLD, preferably according to SEQ ID NO: 2 or a biologically active fragment thereof, for determining the activity of an Mg chelatase and for raising antibodies.

Another object of the invention are methods of generating a protein with the function of a plant Mg chelatase subunit CHLD in a recombinant host cell, wherein this host cell is transformed with a nucleic acid molecule according to the invention encoding a plant Mg chelatase subunit CHLD or an active fragment thereof, preferably that a DNA sequence encoding a protein with the function of an Mg chelatase or a biologically active fragment thereof is inserted into an expression cassette suitable for the host cell, that the resulting expression cassette is inserted in a suitable manner into a vector suitable for the host cell, that a suitable host cell is transformed with the resulting vector and that the thus transformed host cell is grown in a suitable medium and that the protein which is produced by said host cell and which has the function of a plant Mg chelatase subunit CHLD is isolated in a suitable manner from the culture medium or from the host cell.

Yet another object of the present invention is a method of generating a protein with the function of a plant Mg chelatase or a biologically active fragment thereof in a recombinant host cell, wherein this host cell is transformed with a nucleic acid molecule according to the invention encoding a plant Mg chelatase or an active fragment thereof, preferably that a DNA sequence encoding a protein with the function of an Mg chelatase is inserted into an expression cassette which is suitable for the host cell; that the resulting expression cassette is inserted in a suitable manner into a vector suitable for the host cell, that a suitable host cell is transformed with the resulting vector; and that the host cell thus transformed is grown in a suitable medium and that the protein which is produced by said host cell and which has the function of a plant Mg chelatase is isolated in a suitable manner from the culture medium or the host cell.

In this sense, the present invention relates to the generation of plant Mg chelatase subunit CHLD and of plant Mg chelatase by genetic engineering. For example, to generate the proteins according to the invention in a host organism, the DNA sequences according to the invention can be cloned into an expression cassette which is suitable for heterologous expression of the structural gene in the selected host organism.

Examples of CHLD-encoding DNA sequences which are suitable for this purpose are the following: plant chlD cDNA, plant chlD cDNA molecules whose sequence has been altered by customary methods, but also synthetic DNA sequences which have been derived from plant chlD cDNA or plant CHLD protein or fragments thereof and which allow expression of a biologically active Mg chelatase CHLD. Moreover, it may be desired to introduce, into the expression cassette, specific regulatory sequences, for example promoters, operator sequences, enhancers, terminators, signal sequences, 5′- and 3′-untranslated sequences, or sequences encoding suitable fusion proteins. The use of regulatory sequences is generally customary technology, which can be varied within wide limits depending on the expression strategy. The resulting chlD expression cassette, provided with the necessary regulatory elements within the correct reading frame of the chlD structural gene, can be inserted into an expression vector with which the host organism selected can be transformed. Suitable expression strategies for generating recombinant proteins, and corresponding expression vectors, are generally known for host organisms such as, for example, Escherichia coli, insect cells and plant cells.

In general, the term “vector” refers to a suitable vehicle which is known to those skilled in the art and which allows the targeted transfer of a single- or double-stranded nucleic acid molecule into a host cell, for example into a DNA or RNA virus, a virus fragment, a plasmid construct which can be suitable for transferring nucleic acids into cells in the presence or absence of regulatory elements, metal particles as they can be employed, for example, in the particle-gun method, but it may also include a nucleic acid molecule which can be directly introduced into a cell by chemical or physical methods.

The recombinant organism obtained by stable or transient transformation for example with a chlD expression cassette can be used for obtaining recombinant Mg chelatase, preferably CHLD protein, or cell fractions containing CHLD. However, the recombinant organism may also directly be a component of an analytic test system.

It is also possible for the chlD structural gene together with the structural genes encoding the two other known subunits of Mg chelatase, namely CHLH (SEQ ID NO: 6) and CHLI (SEQ ID NO: 5) to be introduced into a host organism with the aid of conventional molecular-genetic processes, resulting in expression of a functional Mg chelatase in this host organism.

A preferred expression system is, for example, the use of bakers yeast (Saccharomyces cerevisiae) as host organism. Vectors which may be used are all known yeast vectors which have the suitable expression signals such as promoters, and suitable selection markers such as resistance genes or genes which complement for an auxotrophism.

The use of plant Mg chelatase is essentially based on its activity as a heterooligomeric enzyme complex, and this activity is directly linked to the presence of the subunit CHLD. Providing functionally intact plant Mg chelatase allows biochemical reactions to be performed not only in vitro (for example cell-free test system of Mg chelatase), but also in vivo, for example in uni- or multicellular recombinant organisms or cell cultures, in particular yeasts, bacteria, algae, insect cells or plants, and is therefore not limited to phototrophic organisms.

On the one hand, these reactions can be exploited for generating Mg-tetrapyrroles, on the other hand, these biochemical reactions can be used for determining, in a test system, the effect of chemical compounds, but also of heterogenous substance mixtures, relative to the function of Mg chelatase.

The present invention therefore furthermore relates to a method of determining the interaction between plant Mg chelatase subunits, which comprises transforming a host cell with a DNA sequence encoding a protein with the function of a subunit CHLD or with a biologically active fragment thereof and at least with one DNA sequence encoding a further subunit of Mg chelatase in such a manner that the interaction of the Mg chelatase gene products leads to a signal which can be measured directly or indirectly, qualitatively or quantitatively, preferably by activating a marker gene.

The method is suited for finding specific inhibitors or activators of plant Mg chelatase, allowing, inter alia, substances to be identified which have a potential herbicidal, growth-inhibitory or -enhancing or phytosanitary action.

The principle of these cellular (in-vivo) test systems is based on the fact that a recombinant organism which has been transformed with structure genes of plant Mg chelatase CHLD and which functionally expresses one or both of the other subunits of plant Mg chelatase indicates an essential function of one or more subunits in a manner which allows substances to be found which influence this function.

One function of Mg chelatase subunits consists in interacting with each other. This interaction leads to the formation of an enzyme complex and is a prerequisite for the enzyme function of Mg chelatase.

The present invention therefore relates to a test system which allows interaction of Mg chelatase subunit CHLD with one or two other subunits of the enzyme to be detected, and quantitatively determined, in vivo.

For example, interaction of subunit CHLD with the subunit CHLI can be measured in such a way that both subunits are linked with two other polypeptides or proteins in such a way that interaction of the subunits directly or indirectly causes a reaction in the cell which can be detected qualitatively and/or quantitatively.

Polypeptides which are suitable for this purpose are, for example, domains or subunits of regulatory proteins, for example from cellular signal transduction or transcription processes whose function is mediated via a protein-protein interaction.

Suitable are, for example, regulatory proteins of DNA transcription where the protein-protein interaction of two or more subunits can be detected with the aid of a reporter gene, so that the gene product of the reporter gene is formed only when two or more of the abovementioned subunits interact with each other.

To generate such a test system, for example one of the two Mg chelatase subunits to be tested can be fused to the DNA binding domain, while the other Mg chelatase subunit is fused to the transcription-activating domain of the regulatory protein. It is irrelevant here which of the two proteins is fused to which of the two domains. Thus, CHLD may be linked to the DNA binding domain and CHLI to the transcription-activating domain. This linkage can be mediated via the interaction of a further auxiliary factor, for example a protein-protein interaction or else protein-ligand interaction such as, for example, streptavidin-biotin. However, a covalent linkage, for example by fusion of encoding regions of genes in question, is preferred.

An example of such a regulatory protein is the gene product of the Saccharomyces cerevisiae GAL4 gene.

A host organism, into whose genome an indicator gene with the relevant promoter had already been integrated, is transformed with the abovedescribed fusion genes of Mg chelatase subunits. Such a host organism is distinguished by the fact that it expresses all functions of the transcription system used, with the exception of the regulatory protein intended to be expressed as recombinant protein. If a microorganism is used as the host organism, the recombinant organism can subsequently be isolated and multiplied as required using customary methods of microbiology and is therefore unlimited in its availability for use in the test system. Host organisms which are preferably used are therefore microorganisms which can be transformed and grown readily, such as, for example, yeast species or E. coli.

Furthermore, a promoter suitable for the transcription system is linked to a structural gene which encodes an indicator protein. This recombinant gene is distinguished by the fact that activation of the promoter directly or indirectly leads to expression of the indicator protein. Such an indicator protein is distinguished by the fact that it leads, in the recombinant organism, to a change which can be detected readily, for example by catalyzing pigment formation or a chemoluminescence reaction, by being colored or fluorescent itself, or by contributing to the growth of the microorganism.

The use of the described system in which transcription of an indicator gene is activated has the advantage over the growth test described further below In which a mutant is complemented with an Mg chelatase subunit that the former allows a more specific signal relative to an inhibitor of the enzyme or of an enzyme function, while the latter test first requires that cell growth inhibitors with a site of action other than Mg chelatase be excluded by control experiments. Thus, the present invention prefers the use of an indicator gene system to the growth test.

An example to be mentioned of such a system which puts into reality the abovementioned interactions with the aid of regulatory proteins of the transcription system is, in particular, the Matchmaker™ two-hybrid system by Clontech, Palo Alto, Calif., USA, Catalog No. #K 1605-1, 1995/96, which is hereby incorporated by reference.

To this end, plasmids are generated which cause the expression, in yeast cells, of CHL D, H and I as fusion proteins with the GAL4 binding domain and the GAL4 activation domain. To construct the expression plasmids, the DNA sections which encode the three Mg chelatase subunits are amplified by means of polymerase chain reaction (PCR).

The templates used for amplification are the plasmids which contain the cDNA of the genes in question. The PCR primers used are synthetic oligonucleotides which are distinguished by the fact that the sense primer shows homology to the 5′ end, while the antisense primer shows homology to the 3′ end of the gene in question. The primers may also be provided with restriction sites which facilitate cloning into the vector plasmid.

The preferred vectors used are the shuttle plasmids of the MATCHMAKER™ two-hybrid system (Clontech): pGBT9 and pAS2 each contain a gene which encodes the GAL4 activation domain. pGAD424 and pACT2 each contain a gene which encodes the GAL4 DNA binding domain. pGBT9 and pGAD424, and pAS2 and pACT2, respectively, are in each case used together in a system. pGBT9 and pGAD424 differ from pAS2 and pACT2 by the fact that the abovementioned genes are more weakly expressed in the first two cases than in the last two cases.

Each of the subunits encoding Mg chelatase are cloned individually into the vectors. Recombinant plasmids are introduced into E. coli by means of transformation, multiplied and isolated. In this manner, all 12 possible combinations of all three subunits with all four vectors are performed. The recombinant plasmids are termed as follows:

Gene CHL D in vector pAS2 was termed pCBS1148

Gene CHL D in vector pACT2 was termed pCBS1149

Gene CHL D in vector pGBT9 was termed pCBS1150

Gene CHL D in vector pGAD424 was termed pCBS1151

Gene CHL H in vector pAS2 was termed pCBS1152

Gene CHL H in vector pACT2 was termed pCBS1153

Gene CHL H in vector pGBT9 was termed pCBS1154

Gen CHL H in vector pGAD424 was termed pCBS1155

Gene CHL I in vector pAS2 was termed pCBS1156

Gene CHL I in vector pACT2 was termed pCBS1157

Gene CHL I in vector pGBT9 was termed pCBS1158

Gene CHL I in vector pGAD424 was termed pCBS1159

Then, a reporter yeast strain of the two-hybrid system is transformed with in each case two plasmids. A preferably used reporter yeast strain is a strain which expresses, as indicator protein, a GAL4-inducible beta-galactosidase, preferably strain SFY526 by Clontech. The two plasmids are selected in such a way that in each case one encodes a subunit in fusion with the DNA binding domain while the other encodes a subunit in fusion with the activation domain. Yeast transformation is performed for example by the method of Klebe et al., 1983, Gene, 25, 333-341. To determine induction of the indicator gene, a yeast transformed with both plasmids is grown on solid medium and the activity of beta-galactosidase is determined after blotting onto filters using the method of Breedon and Nasmytl (Breedon, L. and Nasmyth, K. (1985) Regulation of the yeast HO gene. Cold Spring Harbor Symp. Quant. Biol. 50, 643-650).

Alternatively, the transformed yeast is grown in liquid medium and the beta-galactosidase activity is determined in yeast crude extract after cell disruption, using the method of Munder and Fürst (Munder, T. and Fürst, P. (1992). Mol. Cell. Biol. 12, 2091-2099).

To test substances, the recombinant organism is first grown in a suitable medium. The recombinant cells are then incubated together with the substance to be tested. The incubation conditions are selected in such a way that clearly measurable induction of the indicator gene takes place during the incubation time without the addition of substances to be tested. This can be effected for example by the incubation time extending over part of the growth phase.

Alternatively, induction may also be effected by expressing the proteins which play a role in transcription activation, with the aid of an inducible promoter. Induction of the indicator gene can be detected using customary detection reactions. If the indicator gene in the test system is activated, the consequence of gene activation is that a greater amount of reporter protein is formed in the host cells, than without activation of the indicator gene. The test system is preferably selected in such a way that the reporter protein is produced in at least twice the amount. Increase in production of the reporter protein by a factor of at least 10 is especially preferred. If the reporter protein is an enzyme, the enzyme activity can be determined by customary measuring methods, for example by colorimetry, fluorimetry or luminometry. For this purpose, intact cells or extracts therefrom of various degrees of purification may be incubated with a suitable chromogenic enzyme substrate. Incubation with the substrate can be affected as early as during incubation with the substance to be tested, or else afterwards.

If a chemical compound interferes with this interaction between the subunits, for example by binding to an “interface region” of the subunit, this compound inhibits the oligomerization process. Thus, this compound can reduce the amount of enzymatically active Mg chelatase in an organism and thus also have a potential herbicidal action. Thus, finding herbicidal compounds which inhibit the formation of the active Mg chelatase enzyme complex is also possible via the present test system.

To detect specific interaction of the substance with a protein of Mg chelatase, the substance must meet the following conditions:

1) In recombinant cells, a significantly lower induction of the indicator gene must take place after the substance has been added than in cells without substance added.

2) In an analogous transcription system (control system) in which the interaction of the Mg chelatase subunits was replaced by a different interaction, inhibition of the indicator reaction must only be marginally less or not measurable after addition of the substance. Any type of linkage, for example also covalent bonds of proteins, may be used as the other interaction.

If a substance meets the above conditions, it can be examined for its action in other test systems, for example in enzymatic tests with Mg chelatase, in plant cell cultures or in herbological tests on intact plants.

Moreover, an Mg chelatase gene can be introduced into a specific mutant of such an organism which is not capable of growth without function of the Mg chelatase in question, such as, for example, a photoautotrophic microorganism in which one or more Mg chelatase subunits have lost their function by mutation. The use of such a mutant has the particular advantage that growth of the recombinant organism in a suitable medium can thus be taken as a measure for the function of Mg chelatase and can thus quantitatively describe the effect of test substances on Mg chelatase. The growth test is distinguished by particularly simple handling and a rapid turnover of substances.

To this end, the plant chlD gene can be introduced into a mutant of an organism which is characterized in that it has an altered phenotype or is not capable of growth under certain culture conditions without function of the Mg chelatase, preferably without the function of the CHLD protein. Such a mutant can be generated for example by first altering, in a microorganism which requires the activity of an Mg chelatase for phototrophic growth, the structural genes for its own Mg chelatase in such a way that the functional enzyme with Mg chelatase activity is no longer formed, or not in sufficient amounts, so that the resulting organism is no longer capable of phototrophic growth. Furthermore, the plant structural genes chlI and chlH are expressed in this mutant. By additionally expressing the plant chlD structural gene, a functional plant Mg chelatase can be formed in the genetically engineered organism, and this plant Mg chelatase reimparts to this organism the capability of phototrophic growth. The growth of such a recombinant organism is then a direct measure of the plant Mg chelatase activity. A growth test with this recombinant organism makes it possible to determine whether the plant Mg chelatase is inhibited by a chemical compound added to the culture medium. To this end, the genetically engineered organism is grown in culture media under phototrophic culture conditions with and without the compound to be investigated. The chemical compound to be investigated is preferably employed in concentrations between 10⁻⁹ M and 10⁻³ M, especially preferably in concentrations between 10⁻⁷ M and 10⁻⁴ M.

To detect specific inhibition of the Mg chelatase by a chemical compound to be investigated and to exclude other modes of action as a cause for growth inhibition, the compound must meet the following criteria:

1) Growth of the genetically engineered organisms must be significantly poorer in the presence of the compound than in culture medium without the compound. 2) The compound must not significantly reduce growth of the same organisms under heterotrophic conditions.

Like growth, an altered phenotype of a suitably altered organism may also be used as indicator for catalytic activity of plant Mg chelatase. Various pigments of phototrophic microorganisms are made starting from the reaction product of Mg-chelatase, Mg-protoporphyrin. A recombinant microorganism constructed as described above which contains, instead of its own Mg chelatase, a recombinant plant Mg chelatase containing the recombinant CHLD protein, is only capable of forming its Mg-protoporphyrin-derived pigments when the plant Mg chelatase has sufficient activity. The pigmentation of such a recombinant organism is then a direct measure of plant Mg chelatase activity. Using this recombinant organism, analysis of the pigment composition makes it possible to determine whether the plant Mg chelatase is inhibited by a chemical compound which is added to the culture medium. To this end, the genetically engineered organism is grown in culture media with and without the compound to be investigated under culture conditions under which pigments synthesized from Mg-protoporphyrin are formed. The chemical compound to be investigated is preferably employed in concentrations between 10⁻⁹ M and 10⁻³ M, especially preferably in concentrations between 10⁻⁷ M and 10⁻⁴ M. To detect specific inhibition of the Mg chelatase by a chemical compound to be investigated and to exclude other modes of action as a cause for altered pigmentation, the compound must meet the following criteria:

1. The genetically engineered organisms show significantly lesser amounts of pigments formed starting from Mg-protoporphyrin, or an altered pigment composition, in the presence of the compound than in culture medium without the compound.

2. The compound must not significantly change pigment formation of the non-genetically-engineered starting organism under the same culture conditions.

If both criteria are met, it can be assumed that the compound examined is a specific inhibitor of plant Mg chelatase. If only condition 1, but not condition 2 is met, an enzymatic reaction of Mg chelatase makes it possible to determine whether the compound examined is a specific inhibitor of plant Mg chelatase.

If a substance meets the abovementioned conditions, it can be examined further for its action in the plant with or without further biochemical examinations directly either on intact plants or on suitable plant organs. The customary herbological and physiological methods may be used for this purpose. An essential prerequisite for various uses such as, for example, the generation of a biochemical test system for determining a protein function, is that the protein to be investigated can be obtained in the functional state and as pure as possible, i.e. free of interfering activities. As is the case with all cell proteins, this can be effected in the case of a plant Mg chelatase by isolating the individual subunits from plant tissue with the aid of customary protein purification processes. The present invention states that a functional plant Mg chelatase contains, besides the subunits CHLH and CHLI, the subunit CHLD, whose sequence, as an example, is given for Nicotiana tabacum in SEQ ID NO: 2. Isolation of a heterooligomeric protein whose activity is, moreover, also bound to a membrane fraction of the plant cell, as is the case with Mg chelatase, is generally considerably more difficult than isolation of a soluble or homooligomeric, or monomeric, enzyme since the protein loses its enzymatic activity in the course of the purification process, and this can subsequently only be restored by complicated methods. In addition, the enzymes of chlorophyll biosynthesis are formed as a function of the type of differentiation and of the developmental state of the cell. Moreover, these proteins, as is also the case with Mg chelatase, only represent a small part of the total cell protein so that particularly high concentration factors would have to be achieved when isolating them from plant tissue.

Due to the difficulties described, it is preferred according to the invention to use the Mg chelatase-encoding nucleic acid molecules for generating the recombinant plant Mg chelatase, especially preferably the recombinant Mg chelatase complex composed of the proteins CHLD, CHLH and CHLI. The recombinant plant CHLD protein may be very especially preferably mentioned.

Plant Mg chelatase generated by means of genetic engineering, preferably CHLD protein generated by means of genetic engineering, can be purified using a multiplicity of standard methods. Whether a method is suitable depends in each case on the host organism used, the expression strategy and other factors which are known to those skilled in the art of expression and purification of the recombinant proteins. In order to purify the recombinant protein, it may also be fused to other peptide sequences by altering its gene sequence in the expression cassette in a suitable manner. Fusion components to be used preferably are peptides or proteins which, as C- or N-terminal fusions, impart to the recombinant Mg chelatase subunits an affinity with certain column materials, or which allow the expression product to be located at a specific site within or outside of the cell, for example by means of transit or signal peptides or transit or signal sequences. Such fusions must not affect the function of Mg chelatase or must be capable of being cleaved off, for example by incorporating suitable protease cleavage sites. Examples which may be mentioned of fusion components are oligohistidine tails, the Strep-Tag™, glutathione-S-transferase (GST) or the maltose-binding protein (MalE), without this application being limited to the fusion components given by way of example or fragments thereof. To purify an active Mg chelatase complex, it may also suffice if only one of the subunits existing in the complex is fused to such a peptide or protein. Moreover, it is also possible to raise specific antibodies starting from already small amounts of Mg chelatase generated by a recombinant technology, preferably of the CHLD protein, and with the aid of these antibodies Mg chelatase or Mg chelatase subunits can be isolated from recombinant organisms or else from plants.

Generation of Mg chelatase, preferably of the CHLD protein, by means of genetic engineering, and their purification, allow for the first time a plant enzyme with Mg chelatase activity to be obtained in various degrees of purity, up to the pure Mg chelatase complex, composed of the subunits CHLD, CHLH and CHLI, or else the pure CHLD protein. A possible application for isolated or concentrated Mg chelatase is, for example, its use in biochemical test systems for determining the enzyme function.

The present invention furthermore relates to a method of determining the activity of plant Mg chelatase, where a protein is combined with a CHLD function with the gene products of the DNA sequences encoding for Mg chelatase subunits I and H in such a manner that the enzymatic activity of the Mg chelatase gene products leads to a directly or indirectly, qualitatively or quantitatively measurable signal.

The inhibitory effect of a chemical compound on Mg chelatase can be measured via a reduction in the typical catalyst activity of the enzyme or the complete inactivation of Mg chelatase in the presence of this compound. To this end, the plant Mg chelatase, composed of subunits CHLD, CHLH and CHLI, is incubated in a suitable reaction buffer together with its substrates protoporphyrin, ATP and Mg²⁺.

By way of preferred reaction conditions, there may be mentioned a reaction buffer with a pH of between pH 4 and pH 11, preferably 5-10, in particular 6-9, and reaction temperatures between 2 and 50° C., preferably 10-40° C. and the like.

Quantification of the enzyme inhibition can be effected by a simple comparison of the catalytic activity of Mg chelatase in the absence and presence of the chemical compound to be investigated.

To determine the Mg chelatase activity, a variety of biochemical measuring methods may be employed, by means of which either the formation of the reaction products of the Mg-chelatase-catalyzed reaction is measured, for example, Mg-protoporphyrin, ADP or phosphate, or else a decrease in concentration of the enzyme substrates of Mg chelatase is measured, for example protoporphyrin, ATP or Mg²⁺. A large number of standard methods for determining Mg chelatase activity are available to those skilled in the art of performing enzyme tests. Methods which may preferably be mentioned are those for measuring the conversion rate of protoporphyrin to Mg-protoporphyrin in the reaction batch by measuring the different fluorescence properties of Mg-protoporphyrin and protoporphyrin (for example Walker et al., Plant Physiol. Biochem. 30:263-269 (1992)) or by photometric analysis of the altered absorption resulting from the conversion of protoporphyrin to Mg-protoporphyrin (for example Gorchein, Biochem. J. 299:869-874 (1991)) or by quantifying protoporphyrin or Mg-protoporphyrin by HPLC analysis. Determining the ATPase activity of Mg chelatase may be mentioned as a further preferred measuring method, and here it is possible to detect, for example, the formation of phosphate by a phosphate detection, for example by the methods based on the system described by Fiske et al., J. Biol. Chem 66:375-400 (1925). In addition, the inhibitory type of a chemical compound relative to plant Mg chelatase can be determined by varying the concentrations of the substrate and the inhibitory substance, using customary biochemical methods.

Instead of using purified Mg chelatase, it is also possible to use intact cells of the recombinant organism which gives recombinant expression of the three Mg chelatase subunits CHLD, CHLH and CHLI, or protein-containing extracts from this organism, or Mg-chelatase-containing fractions from this organism which have been concentrated to different degrees. Yeast cells may be mentioned as the preferred recombinant host organism for this purpose. Alternatively, it is also possible to use a functional Mg chelatase containing the subunits CHLD, CHLH and CHLI which has been isolated from plant tissue or plant cell cultures.

If, with the aid of the biochemical or cell-biological test systems, it is found that a chemical compound inhibits plant Mg chelatase in vitro, this compound can be investigated directly for its action on plants, either on intact plants or on suitable parts of plants. To this end, a multiplicity of conventional herbological and physiological methods are available to those skilled in the art assessing active substances on plants.

Moreover, the Mg chelatase generated by means of genetic engineering, preferably an Mg chelatase complex with the subunit CHLD which has been generated by means of genetical engineering, or else the free subunit CHLD, can also be used for example for elucidating the spatial structure of the plant enzyme. Generally known methods such as, for example, X-ray structural analysis of protein crystals or NMR spectroscopy may be used for elucidating the spatial structure. The structure information on Mg chelatase, preferably on the subunit CHLD, can be used, for example, for rationally designing novel Mg chelatase inhibitors, and thus potential herbicides.

The disclosures in German patent application 197 17 656.9 from which this application claims priority, and in the abstract accompanying this application are incorporated herein by reference.

The examples which follow are intended to illustrate the invention in greater detail and are in no way to be understood as a limitation:

Standard methods such as DNA and RNA isolation, sequence analysis, restriction, cloning, gel electrophoresis, radio labeling, Southern and Northern blot were carried out by conventional methods (Sambrook at al., 1989 Molecular Screening: A laboratory manual 2^(nd) ed. Cold Spring Harbour Laboratory Press N.Y. USA; Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467).

EXAMPLE 1 Cloning a Tobacco cDNA Which Encodes the Mg Chelatase Subunit CHLD

To identify the tobacco chlD cDNA, the peptide sequences VDASGS (SEQ ID NO: 3), TDGRGN (SEQ ID NO: 4) and AKGAVM (Seq. ID NO: 7) were selected which correspond to the protein sequences derived from the chlD gene of Synechocystis PCC6803 (Jensen et al. (1996) J. Biol. Chem. 271, 16662-16667) to prepare the following mixture of DNA primer sequences:

ga(c/t)gt(a/c/t/g)ga(g/a)aa(a/g)(t/a)c(a/c/g/t)gt(a/c/g/t) (SEQ ID NO: 8)

at(a/g)tt(a/c/t/g)cc(a/c/t/g)cg(a/c/t/g)cc(a/g)tc(a/c/t/g)g (SEQ ID NO: 9)

gc(a/c/t/g)aa(a/g)gg(a/c/t/g)gc(a/c/t/g)gt(a/c/t/g)atgc (SEQ ID NO: 10)

These primers were used in a polymerase chain reaction (PCR) to amplify the genomic DNA from the Synechocystis PCC6803: 2 cycles with in each case 1 min. 94° , 2 min. 45° C., 3 min. 72° C., then 28 cycles in each case 30 sec. 94° C., 90 sec. 60° C., 2 min. 72° C.

The PCR resulted in a DNA fragment of approx. 270 bp which was cloned into the cloning vector pCRTMII (Invitrogen, San Diego) and isolated for further handling by means of a restriction digest.

The isolated and ³²P[dCTP]-labeled fragment of approx. 270 bp in length was used as a hybridization probe to screen a lambda ZAP II cDNA library of tobacco (Nicotiana tabacum SR1, Stratagene) following the instructions.

The DNA present after a cDNA library screening in the phagemid pBluescript SK was isolated and sequenced. The chlD cDNA sequence identified and shown in SEQ ID NO: 1 contains an open reading frame 2274 nucleotides in length, and untranslated regions on the 5′ and 3′ termini.

Genomic tobacco DNA was hybridized with radio labeled DNA of the CHLD gene. To this end, the CHLD gene was excised from the phagemid pBluescript SK using EcoRI and HindIII. The resulting two fragments of sizes 2069 bp and 426 bp were radio labeled using ³²P(dCTP).

EXAMPLE 2 Generation of chlD Antisense Tobacco Plants

The tobacco cDNA fragment for CHLD which was obtained according to Example 1 in pBluescript SK—was excized from the vector pBluescript SK-using KpnI and XbaI and ligated into the binary vector BinAR (Höfgen and Willmitzer, Plant Science (1990) 66, 22-230) which had been cleaved with KpnI and XbaI.

First, E. coli strain DH 5 α was transformed with the construct and then Agrobacterium tumefaciens strain GV 2260. Using the leaf-disk transformation method (Horsch et al., Science 228, 1229-31), young tobacco leaf disks were incubated with the agrobacteria, and the CHLD antisense gene was stably-integrated into the plant genome.

EXAMPLE 3 Generation of the chlD Sense Tobacco Plants

To generate the chlD sense tobacco plants, the chlD cDNA was excized from p Bluescript SK-using SmaI and XbaI and cloned into the BinAR vector after having been subjected to restriction digest. To stably integrate it into the tobacco genome, the method described in Example 2 was chosen.

EXAMPLE 4 Analysis of the Transgenic Tobacco Plants

67 sense plants and 56 antisense plants were isolated. The transgenic plants with antisense or sense genes for CHLD showed gradually different chlorophyll deficiencies. Chlorotic leaves showed a variety of variation patterns. There were uniformly decolorized, yellowish-green leaves, leaves with differently pigmented spots, and leaves with white areas along the leaf veins and green intercostal areas. As the leaves aged, they lost chlorophyll. Plants with reduced chlorophyll content also showed reduced growth (see FIG. 1a to 1 d).

Moreover, the stable integration of the sense or antisense chlD genes into the genome of tobacco plants was confirmed by Southern blot analysis, and the mRNA content of the chlD transcript was determined by Northern blot analysis. In the antisense plants, the content is reduced in comparison with the wild type. In comparison with the wild type, chlD sense plants only have slightly elevated chlD mRNA contents.

The Mg chelatase activity was determined for transgenic and wild-type plants by the method of Lee et al. (1992, Plant Physiol. 99, 1134-1140). The sense and antisense plants always show reduced enzyme activities.

The phenomenon of the reduced enzyme activities in the supposedly CHLD-overexpressed transformants can be explained by negative dominance. An oversupply of the CHLD subunit causes a disruption of the finely regulated construction of the Mg chelatase enzyme complex.

The protoporphyrin IX and chlorophyll content was determined in transgenic and wild-type plants (see Table 2). It must be emphasized that the transformants with sense or antisense CHLD mRNA show, as a rule, protoporphyrin contents in their young leaves tested (leaf 3, 5 and 7) which are up to 3-5 times higher than those of the wild-type plants.

The determination of the chlorophyll content confirms the macroscopic phenotype. In individual plants, the content was reduced by up to 25%.

TABLE 2 Relative protoprophyrin IX and chlorophyll content in sense and antisense plants Plant Leaf Protoporphyrin IX [%] Chlorophyll [%] SNN 3 100 100 5 100 7 100 100 AS7 3 584 25 5 542 7 157 28 AS9 3 135 50 5 120 — 7 — 49 AS13 3 160 78 5 102 — 7 115 78 AS18 3 374 74 5 470 — 7 295 80 AS21 3 231 95 5 188 — 7 98 101 S1 3 189 78 5 262 — 7 297 80 S20 3 423 32 5 293 — 7 144 25 S22 3 189 117 5 151 — 7 115 94 S29 3 298 25 5 265 — 7 137 26 S38 3 257 120 5 127 — 7 — 107 SNN = Wild type S = Sense As = Antisense

EXAMPLE 5 Construction of Plasmids for Expressing CHL D, H and I as Fusion Proteins With the GAL4 Binding Domain and the GAL4 Activation Domain

To construct the expression plasmids, the DNA encoding the three Mg chelatase subunits was amplified by means of polymerase chain reaction (PCR).

To amplify the chlD gene, 100 ng of plasmid pNTCHLD were used as template. The following two oligonucleotides were used as PCR primers: 5′-TGA CCC GGG GGT AGT GGA ACC TGA AAA ACA ACC-3′ (SEQ ID NO: 10) as sense primer with SmaI restriction site, and either 5′-GGC GAA TTC TCA AGA TTC CTT TAA TGC AGA TAA-3′ (SEQ ID NO: 12) with EcoRI restriction site or 5′-GCG GTC GAC TCA AGA TTC CTT TAA TGC AGA-3′ (SEQ ID NO: 13) with SaII restriction site as antisense primer.

To amplify the CHLH gene, 100 ng of plasmid pNTCHLH were used as template. The following two oligonucleotides were used as PCR primers: 5′-GCT GAT ATC GGC TAT TGG CAA TGG TTT ATT CAC-3′ (SEQ ID NO:14) with EcoRV restriction site as sense primer and 5′-GCG TCG ACA TTT ATC GAT CGA TTC CCT CAA-3′ (SEQ ID NO: 15) with SaII restriction site as antisense primer. To amplify the chlI gene, 100 ng of plasmid PNTCHLI were used as template. The following two oligonucleotides were employed as PCR primers: 5′-CAG CCC GGG GGG TCC ACT ACT AGG-3′ (SEQ ID NO: 16) with SmaI restriction sites sense primer and 5′-CAG GTC GAG GCA CAG TAC AAA GCC-3′ (SEQ ID NO: 17) with SalI restriction site as antisense primer.

The shuttle plasmids of MATCHMAKER™ two-hybrid system (Clontech) were used as vectors: pGBT9 and pAS2 each contain a gene which encodes the GAL4 activation domain. pGAD424 and pACT2 each contain a gene which encodes the GAL4 DNA binding domain. pGBT9 and pGAD424, and pAS2 and pACT2, respectively, are in each case used together in a system. pGBT9 and pGAD424 differ from pAS2 and pACT2 by the fact that the abovementioned genes are more weakly expressed in the first two cases than in the last two cases.

To clone chlD into vector pACT2, the gene was amplified by means of the sense primer with SmaI cleavage site and the antisense primer with EcoRI cleavage site and subsequently ligated with the SmaI- and EcoRI-cut vector.

To clone chlD into in each case one of vectors pAS2, pGBT9 and pGAD424 the gene was amplified by means of the sense primer with SmaI cleavage site and the antisense primer with SaII cleavage site and subsequently ligated with the SmaI- and SaII-cut vector.

To clone in each case one of genes chlH and I into in each case one of vectors pAS2, pGBT9 and pGAD424, the genes were amplified in each case by means of the sense and the antisense primer and subsequently ligated with the vectors cut in each case with SmaI and SaII.

To clone in each case one of genes chlH and I into vector pACT2, the genes were amplified by means of the sense and the antisense primer and subsequently ligated with the SmaI- and XhoI-cut vector. The ligated DNA vias introduced into E. coli DH 5a by means of transformation, and transformed clones were selected on agar medium with 100 μg ampicillin/ml. Plasmid DNA was isolated from the recombinant bacteria and tested for identity by means of restriction analysis.

All 12 possible combinations of all three subunits with all four vectors were made in this manner.

EXAMPLE 6 Determining the Interaction Between Mg Chelatase Subunits by Means of the Two-hybrid System in Yeast

β-Galactosidase liquid assay in yeast (Munder and Fürst, 1992, Mol.Cell.Biol. 12, 2091-2099) using strain SFY526+pAS2-chlD+pACT2-chlI as example.

Competent yeast cells strain SFY 526 (Harper et al., 1993, Cell, 75, 805-816) were made by the method of Klebe (Klebe et al., 1983, Gene, 25, 333-341) and transformed with the plasmids given in Example 2. SFY 526 was transformed with all plasmids individually and in all possible combinations and selected on minimal agar (1.5% agar, 10% YNB glucose) with an addition of amino acids (adenine 20 mg/l; L-histidine.HCl 20 mg/l; L-lysine.HCl 30 mg/l; L-leucine 30 mg/l; L-tryptophane 20 mg/l).

The yeast strain SFY526+pAS2-chlD+pACT2-chlI to be measured was grown in 10 ml of minimal medium (10% of YNB glucose) with an addition of amino acids required (adenine 20 mg/l; L-histidine HCl 20 mg/l and L-lysine HCl 30 mg/l) overnight at 30° C. and 180 rpm in a sterile 50 ml wide-necked flask with metal cap. The optical density (OD₆₀₀) of this overnight culture was recorded (Beckman DU 640 spectrophotometer), and the values measured were between 1.0 and 2.0.

100 μl of this culture were tested for β-galactosidase activity. The total sample volume was approx. 1 ml:

100 μl culture

700 μl Z buffer+mercaptoethanol

50 μl trichloromethane

50 μl 0.1% SDS

A zero-value was examined in parallel:

700 μl Z buffer

50 μl trichloromethane

50 μl 0.1% SDS

Sample and parallel zero value were each vortexed for 30 seconds, and 160 μl of ONPG solution(4 mg o-nitrophenyl-β-_(D)-galactopyranoside on 1 ml of Z buffer+mercaptoethanol) was added to each of them. The samples were then shaken carefully and incubated in a water bath at 30° C. After one hour, the reaction was quenched by adding 400 μl of 1M Na₂CO₃, and the samples were spun for 10 minutes at 13,000 rpm (Heraeus Biofuge fresco, fixed-angle rotor 24). The supernatant was measured at 420 nm against the zero-value (Beckman DU 640 spectrophotometer), and the β-galactosidase activity was calculated:

U=1000 E₄₂₀ (CVt)⁻¹

E₄₂₀ is the extinction at 420 nm, C is the density of the cell suspension (OD₆₀₀), V is the cell suspension volume employed, and t is the incubation time.

The measurement was repeated on two further clones, and the mean was determined.

Solutions required:

Z buffer 1000 ml:

16.1 g Na₂HPO₄x7H₂O

5.5 g NaH₂PO₄x7H₂O

0.75 g KCl

MgSO₄x7H₂O

Z buffer+mercaptoethanol (make freshly)

100 ml of Z buffer+270 μl mercaptoethanol

EXAMPLE 7 Reconstitution Assay Mg Chelatase Activity in Yeast (Willows et al., 1996, Eur.J.Biochem. 235,438-443) Strain SFY526+pAS2-chlD+pACT2-chlH+pSG28

Competent cells of strain SFY526+pAS2-chlD+pACT2-chlH were prepared (Klebe et al.; 1983; Gene, 25, 333-341) and transformed with pSG28 (p423TEF+chlI; p423 TEF: Mumberg et al.; 1995; Gene, 156;119-122). Transformants were selected on minimal agar (10% YNB glucose; 1.5% agar) with an addition of adenine (20 mg/l) and L-lysine.HCl (30 mg/l). 15 ml of a preculture (minimal medium: 10% YNB glucose+20 mg/l adenine+30 mg/l L-lysine HCl) of this strain were grown for 24 hours at 30° C. and 180 rpm in a sterile 50 ml wide-necked flask with metal cap. 150 ml of main culture (minimal medium: 10% YNB glucose+20 mg/l adenine+30 mg/l L-lysine HCl) were inoculated with 7.5 ml of preculture and shaken for 20 hours at 30° C. and 180 rpm using a sterile 500 ml Erlenmeyer flask with cottonwool plug. The cells were sedimented for 5 minutes at 5000 rpm (Sigma 4K 10) and resuspended in 1.5 ml of assay buffer (0.1 M tricine pH 7.9, 0.3 M glycerol, 25 mM MgCl₂, 4 mM DTT). This cell suspension was subsequently disrupted ultrasonically for 3×30 seconds on ice at low ultrasonic strength.

The disrupted cells were sedimented for 15 minutes at 5000 rpm (Sigma 4 K 10) and the supernatant was processed further. The protein content of this yeast extract was determined using the BioRad protein assay by the method of Bradford.

The assay volume was 1 ml. 1 ml of assay buffer contained 1 mg of protein extract of the disrupted yeast culture, 4 mM ATP, 1.5 μM protoporphyrin IX, 50 mM phosphocreatine and 10 U creatinephosphokinase.

This incubation mix was incubated for one hour in the dark at 30° C., and a fluorescence emission spectrum between 500 and 650 nm at an excitation wavelength of 420 nm was then recorded in a Perkin Elmer luminescence spectrometer LS50 B and subjected to HPLC analysis.

17 1 2495 DNA Nicotiana tabacum CDS (40)..(2313) coding sequence for subunit chlD of plant magnesium chelatases 1 catctaaaat cctaaatcaa aaacttcgat gctataaaa atg ggg ttt tgt tca 54 Met Gly Phe Cys Ser 1 5 act tca acc ctc cca caa aca tca cta tcc aat tct caa tct tca aca 102 Thr Ser Thr Leu Pro Gln Thr Ser Leu Ser Asn Ser Gln Ser Ser Thr 10 15 20 ttc ttc aca tac tta aaa cca tgc cca att cta tcc tcc aca tat tta 150 Phe Phe Thr Tyr Leu Lys Pro Cys Pro Ile Leu Ser Ser Thr Tyr Leu 25 30 35 agg ccg gaa cgg cta aaa ttt cgc ctc aga ata agt gcc act gca act 198 Arg Pro Glu Arg Leu Lys Phe Arg Leu Arg Ile Ser Ala Thr Ala Thr 40 45 50 att gat tca cct aat ggc gct gtt gca gta gtg gaa cct gaa aaa caa 246 Ile Asp Ser Pro Asn Gly Ala Val Ala Val Val Glu Pro Glu Lys Gln 55 60 65 cct gag aaa att tcc ttt ggt aga cag tat ttt cct cta gct gct gtt 294 Pro Glu Lys Ile Ser Phe Gly Arg Gln Tyr Phe Pro Leu Ala Ala Val 70 75 80 85 att gga cag gat gct att aaa act gct ctt tta ctt ggg gcc att gac 342 Ile Gly Gln Asp Ala Ile Lys Thr Ala Leu Leu Leu Gly Ala Ile Asp 90 95 100 cgt gag ata gga gga att gca ata tgt ggg aag cgt gga aca gcg aaa 390 Arg Glu Ile Gly Gly Ile Ala Ile Cys Gly Lys Arg Gly Thr Ala Lys 105 110 115 acg tta atg gca cgt gga ttg cat gct att ctg cca cca att gaa gta 438 Thr Leu Met Ala Arg Gly Leu His Ala Ile Leu Pro Pro Ile Glu Val 120 125 130 gtt gtt ggc tca atg gca aat gct gat ccg aac tgt ccc gat gag tgg 486 Val Val Gly Ser Met Ala Asn Ala Asp Pro Asn Cys Pro Asp Glu Trp 135 140 145 gaa gac ggg cta gct gac aga gca gaa tat ggg tct gat ggt aat atc 534 Glu Asp Gly Leu Ala Asp Arg Ala Glu Tyr Gly Ser Asp Gly Asn Ile 150 155 160 165 aag acc cag ata gtt aaa tcc cca ttt gtt cag att ccc ctt ggt gtc 582 Lys Thr Gln Ile Val Lys Ser Pro Phe Val Gln Ile Pro Leu Gly Val 170 175 180 aca gaa gat aga ttg att ggc tct gtt gat gtc gag gag tcc gtg aaa 630 Thr Glu Asp Arg Leu Ile Gly Ser Val Asp Val Glu Glu Ser Val Lys 185 190 195 tct gga acc act gtc ttt caa cca ggc ctc ctc gca gaa gct cat cga 678 Ser Gly Thr Thr Val Phe Gln Pro Gly Leu Leu Ala Glu Ala His Arg 200 205 210 gga gtt cta tat gtt gat gag att aat cta tta gat gaa ggt ata agt 726 Gly Val Leu Tyr Val Asp Glu Ile Asn Leu Leu Asp Glu Gly Ile Ser 215 220 225 aac cta ctt ctg aat gta ttg act gag gga gtc aat att gta gaa aga 774 Asn Leu Leu Leu Asn Val Leu Thr Glu Gly Val Asn Ile Val Glu Arg 230 235 240 245 gag gga atc agc ttt cga cat cca tgc aaa cca cta cta att gct acc 822 Glu Gly Ile Ser Phe Arg His Pro Cys Lys Pro Leu Leu Ile Ala Thr 250 255 260 tat aac cct gaa gag ggt gcg gtt cgt gag cat ctg cta gac cgt att 870 Tyr Asn Pro Glu Glu Gly Ala Val Arg Glu His Leu Leu Asp Arg Ile 265 270 275 gcg att aat tta agt gca gat ctt cca atg agt ttt gac gat cgt gtt 918 Ala Ile Asn Leu Ser Ala Asp Leu Pro Met Ser Phe Asp Asp Arg Val 280 285 290 gca gct gtt gac ata gca aca cgt ttt cag gag tgt agc aat gag gtt 966 Ala Ala Val Asp Ile Ala Thr Arg Phe Gln Glu Cys Ser Asn Glu Val 295 300 305 ttt aaa atg gtg gat gaa gaa aca gac agt gca aaa acc cag ata ata 1014 Phe Lys Met Val Asp Glu Glu Thr Asp Ser Ala Lys Thr Gln Ile Ile 310 315 320 325 ttg gca agg gag tat tta aag gat gtc aca atc agt aga gat caa cta 1062 Leu Ala Arg Glu Tyr Leu Lys Asp Val Thr Ile Ser Arg Asp Gln Leu 330 335 340 aaa tac ttg gtc atg gaa gca att cgt ggt ggc tgc cag ggg cac cga 1110 Lys Tyr Leu Val Met Glu Ala Ile Arg Gly Gly Cys Gln Gly His Arg 345 350 355 gct gaa ctt tat gct gct cgt gta gcc aaa tgc tta gct gcc atc gat 1158 Ala Glu Leu Tyr Ala Ala Arg Val Ala Lys Cys Leu Ala Ala Ile Asp 360 365 370 gga cgt gaa aaa gtt ggt gtt gat gag ctg aaa aaa gct gta gag ctt 1206 Gly Arg Glu Lys Val Gly Val Asp Glu Leu Lys Lys Ala Val Glu Leu 375 380 385 gtc atc ctc cca cgt tca act ata gtt gaa aac cca cca gac cag caa 1254 Val Ile Leu Pro Arg Ser Thr Ile Val Glu Asn Pro Pro Asp Gln Gln 390 395 400 405 aac cag cag cca cct cct ccc cct ccc cct ccc caa aat caa gat tct 1302 Asn Gln Gln Pro Pro Pro Pro Pro Pro Pro Pro Gln Asn Gln Asp Ser 410 415 420 tca gaa gag cag aat gaa gaa gaa gaa aaa gaa gaa gaa gat caa gag 1350 Ser Glu Glu Gln Asn Glu Glu Glu Glu Lys Glu Glu Glu Asp Gln Glu 425 430 435 gat gag aaa gat aga gaa aat gaa cag caa cag cca caa gtc cct gat 1398 Asp Glu Lys Asp Arg Glu Asn Glu Gln Gln Gln Pro Gln Val Pro Asp 440 445 450 gag ttt att ttt gat gcg gaa ggt ggt tta gtg gat gaa aaa ctt ctc 1446 Glu Phe Ile Phe Asp Ala Glu Gly Gly Leu Val Asp Glu Lys Leu Leu 455 460 465 ttc ttt gca caa caa gca caa aga cgc aaa gga aaa gct gga cga gca 1494 Phe Phe Ala Gln Gln Ala Gln Arg Arg Lys Gly Lys Ala Gly Arg Ala 470 475 480 485 aag aag gtc atc ttt tcc gaa gat aga ggt cga tat ata aag cca atg 1542 Lys Lys Val Ile Phe Ser Glu Asp Arg Gly Arg Tyr Ile Lys Pro Met 490 495 500 ctt cca aag ggt cca gtg aag aga ttg gca gtt gat gca act cta aga 1590 Leu Pro Lys Gly Pro Val Lys Arg Leu Ala Val Asp Ala Thr Leu Arg 505 510 515 gca gcg gca cca tat cag aag tta cga aga gca aag gac atc caa aaa 1638 Ala Ala Ala Pro Tyr Gln Lys Leu Arg Arg Ala Lys Asp Ile Gln Lys 520 525 530 act cgc aag gtt tat gta gag aaa act gac atg aga gcc aaa aga atg 1686 Thr Arg Lys Val Tyr Val Glu Lys Thr Asp Met Arg Ala Lys Arg Met 535 540 545 gca cgc aaa gcc gga gct ctg gtg ata ttc gta gtt gac gct agt ggg 1734 Ala Arg Lys Ala Gly Ala Leu Val Ile Phe Val Val Asp Ala Ser Gly 550 555 560 565 agt atg gca ctg aat aga atg cag aat gcc aaa gga gca gca ctt aaa 1782 Ser Met Ala Leu Asn Arg Met Gln Asn Ala Lys Gly Ala Ala Leu Lys 570 575 580 cta ctt gca gag agt tat aca agc aga gat cag gtc tgt atc att ccc 1830 Leu Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Cys Ile Ile Pro 585 590 595 ttc cgc gga gat gct gct gaa gtt ttg ttg cca cct tct agg tca ata 1878 Phe Arg Gly Asp Ala Ala Glu Val Leu Leu Pro Pro Ser Arg Ser Ile 600 605 610 tcg atg gca aga aat cgt ctt gag aga ctt ccc tgt gga ggg ggt tct 1926 Ser Met Ala Arg Asn Arg Leu Glu Arg Leu Pro Cys Gly Gly Gly Ser 615 620 625 ccc ctt gct cat ggg ctt acg acg gca gtt aga gtt gga atg aat gca 1974 Pro Leu Ala His Gly Leu Thr Thr Ala Val Arg Val Gly Met Asn Ala 630 635 640 645 gaa aag agt ggt gat gtt gga cgt atc atg att gtt gca att act gat 2022 Glu Lys Ser Gly Asp Val Gly Arg Ile Met Ile Val Ala Ile Thr Asp 650 655 660 ggt aga gct aac atc tct ctt aaa aga tcc aca gac cct gaa gct gaa 2070 Gly Arg Ala Asn Ile Ser Leu Lys Arg Ser Thr Asp Pro Glu Ala Glu 665 670 675 gct tct gat gca ccc aga cct tct tcc caa gag ctg aag gat gag att 2118 Ala Ser Asp Ala Pro Arg Pro Ser Ser Gln Glu Leu Lys Asp Glu Ile 680 685 690 ctc gag gtg gct ggt aaa ata tac aaa aca gga atg tct ctc ctc gtc 2166 Leu Glu Val Ala Gly Lys Ile Tyr Lys Thr Gly Met Ser Leu Leu Val 695 700 705 ata gat aca gaa aat aag ttt gtt tct act ggt ttt gcg aaa gaa atc 2214 Ile Asp Thr Glu Asn Lys Phe Val Ser Thr Gly Phe Ala Lys Glu Ile 710 715 720 725 gcg aga gta gct caa ggg aag tac tat tat tta cca aat gct tca gat 2262 Ala Arg Val Ala Gln Gly Lys Tyr Tyr Tyr Leu Pro Asn Ala Ser Asp 730 735 740 gct gtg ata tct gca gca aca aag gat gca tta tct gca tta aag gaa 2310 Ala Val Ile Ser Ala Ala Thr Lys Asp Ala Leu Ser Ala Leu Lys Glu 745 750 755 tct tgacctaaac tcgatcgaat taattgtaaa tgttgttttg agtatagatt 2363 Ser attgggagga tataagagct tgcttgataa ttcttatctt ttgttgtact aattgaactt 2423 atttctcaat tatgcaatca gggtaatgaa gattcttttc atttcaaaaa aaaaaaaaaa 2483 aaaggaattc ga 2495 2 758 PRT Nicotiana tabacum 2 Met Gly Phe Cys Ser Thr Ser Thr Leu Pro Gln Thr Ser Leu Ser Asn 1 5 10 15 Ser Gln Ser Ser Thr Phe Phe Thr Tyr Leu Lys Pro Cys Pro Ile Leu 20 25 30 Ser Ser Thr Tyr Leu Arg Pro Glu Arg Leu Lys Phe Arg Leu Arg Ile 35 40 45 Ser Ala Thr Ala Thr Ile Asp Ser Pro Asn Gly Ala Val Ala Val Val 50 55 60 Glu Pro Glu Lys Gln Pro Glu Lys Ile Ser Phe Gly Arg Gln Tyr Phe 65 70 75 80 Pro Leu Ala Ala Val Ile Gly Gln Asp Ala Ile Lys Thr Ala Leu Leu 85 90 95 Leu Gly Ala Ile Asp Arg Glu Ile Gly Gly Ile Ala Ile Cys Gly Lys 100 105 110 Arg Gly Thr Ala Lys Thr Leu Met Ala Arg Gly Leu His Ala Ile Leu 115 120 125 Pro Pro Ile Glu Val Val Val Gly Ser Met Ala Asn Ala Asp Pro Asn 130 135 140 Cys Pro Asp Glu Trp Glu Asp Gly Leu Ala Asp Arg Ala Glu Tyr Gly 145 150 155 160 Ser Asp Gly Asn Ile Lys Thr Gln Ile Val Lys Ser Pro Phe Val Gln 165 170 175 Ile Pro Leu Gly Val Thr Glu Asp Arg Leu Ile Gly Ser Val Asp Val 180 185 190 Glu Glu Ser Val Lys Ser Gly Thr Thr Val Phe Gln Pro Gly Leu Leu 195 200 205 Ala Glu Ala His Arg Gly Val Leu Tyr Val Asp Glu Ile Asn Leu Leu 210 215 220 Asp Glu Gly Ile Ser Asn Leu Leu Leu Asn Val Leu Thr Glu Gly Val 225 230 235 240 Asn Ile Val Glu Arg Glu Gly Ile Ser Phe Arg His Pro Cys Lys Pro 245 250 255 Leu Leu Ile Ala Thr Tyr Asn Pro Glu Glu Gly Ala Val Arg Glu His 260 265 270 Leu Leu Asp Arg Ile Ala Ile Asn Leu Ser Ala Asp Leu Pro Met Ser 275 280 285 Phe Asp Asp Arg Val Ala Ala Val Asp Ile Ala Thr Arg Phe Gln Glu 290 295 300 Cys Ser Asn Glu Val Phe Lys Met Val Asp Glu Glu Thr Asp Ser Ala 305 310 315 320 Lys Thr Gln Ile Ile Leu Ala Arg Glu Tyr Leu Lys Asp Val Thr Ile 325 330 335 Ser Arg Asp Gln Leu Lys Tyr Leu Val Met Glu Ala Ile Arg Gly Gly 340 345 350 Cys Gln Gly His Arg Ala Glu Leu Tyr Ala Ala Arg Val Ala Lys Cys 355 360 365 Leu Ala Ala Ile Asp Gly Arg Glu Lys Val Gly Val Asp Glu Leu Lys 370 375 380 Lys Ala Val Glu Leu Val Ile Leu Pro Arg Ser Thr Ile Val Glu Asn 385 390 395 400 Pro Pro Asp Gln Gln Asn Gln Gln Pro Pro Pro Pro Pro Pro Pro Pro 405 410 415 Gln Asn Gln Asp Ser Ser Glu Glu Gln Asn Glu Glu Glu Glu Lys Glu 420 425 430 Glu Glu Asp Gln Glu Asp Glu Lys Asp Arg Glu Asn Glu Gln Gln Gln 435 440 445 Pro Gln Val Pro Asp Glu Phe Ile Phe Asp Ala Glu Gly Gly Leu Val 450 455 460 Asp Glu Lys Leu Leu Phe Phe Ala Gln Gln Ala Gln Arg Arg Lys Gly 465 470 475 480 Lys Ala Gly Arg Ala Lys Lys Val Ile Phe Ser Glu Asp Arg Gly Arg 485 490 495 Tyr Ile Lys Pro Met Leu Pro Lys Gly Pro Val Lys Arg Leu Ala Val 500 505 510 Asp Ala Thr Leu Arg Ala Ala Ala Pro Tyr Gln Lys Leu Arg Arg Ala 515 520 525 Lys Asp Ile Gln Lys Thr Arg Lys Val Tyr Val Glu Lys Thr Asp Met 530 535 540 Arg Ala Lys Arg Met Ala Arg Lys Ala Gly Ala Leu Val Ile Phe Val 545 550 555 560 Val Asp Ala Ser Gly Ser Met Ala Leu Asn Arg Met Gln Asn Ala Lys 565 570 575 Gly Ala Ala Leu Lys Leu Leu Ala Glu Ser Tyr Thr Ser Arg Asp Gln 580 585 590 Val Cys Ile Ile Pro Phe Arg Gly Asp Ala Ala Glu Val Leu Leu Pro 595 600 605 Pro Ser Arg Ser Ile Ser Met Ala Arg Asn Arg Leu Glu Arg Leu Pro 610 615 620 Cys Gly Gly Gly Ser Pro Leu Ala His Gly Leu Thr Thr Ala Val Arg 625 630 635 640 Val Gly Met Asn Ala Glu Lys Ser Gly Asp Val Gly Arg Ile Met Ile 645 650 655 Val Ala Ile Thr Asp Gly Arg Ala Asn Ile Ser Leu Lys Arg Ser Thr 660 665 670 Asp Pro Glu Ala Glu Ala Ser Asp Ala Pro Arg Pro Ser Ser Gln Glu 675 680 685 Leu Lys Asp Glu Ile Leu Glu Val Ala Gly Lys Ile Tyr Lys Thr Gly 690 695 700 Met Ser Leu Leu Val Ile Asp Thr Glu Asn Lys Phe Val Ser Thr Gly 705 710 715 720 Phe Ala Lys Glu Ile Ala Arg Val Ala Gln Gly Lys Tyr Tyr Tyr Leu 725 730 735 Pro Asn Ala Ser Asp Ala Val Ile Ser Ala Ala Thr Lys Asp Ala Leu 740 745 750 Ser Ala Leu Lys Glu Ser 755 3 6 PRT Synechocystis PCC6803 PEPTIDE (1)..(6) peptide sequence derived from chlD gene of synechocystis PCC6803 3 Val Asp Ala Ser Gly Ser 1 5 4 6 PRT Synechocystis PCC6803 PEPTIDE (1)..(6) peptide sequence derived from chlD gene of synechocystis PCC6803 4 Thr Asp Gly Arg Gly Asn 1 5 5 1579 DNA Nicotiana tabacum misc_feature (1)..(1579) coding sequence for subunit chlI of plant magnesium chelatases 5 cccaaaattc ttcttcttct tcttcactga aaaattctaa acaaaatggc ttcactacta 60 ggaacttcct cttcagcagc agctgcaata ttagcttcta cacccttgtc ttctcgctcc 120 tgtaagcctg ccgttttctc cctcttccct tcttcagggc agagtcaagg gaggaagttt 180 tatggaggga ttagagtccc agttaagaaa gggaggtccc aattccatgt ggcaatttca 240 aatgttgcga cggaaatcaa cctgctcaag aacagggtca gaaacttgct ggaggagagc 300 cagagaccgg tgtatccatt tgcagctata gtgggacaag atgaaatgaa gttatgtctt 360 ttgctgaatg taattgatcc aaagattgga ggtgtgatga taatgggtga taggggaacc 420 gggaagtcca ccacggttag atctttggta gatttacttc ctgaaatcaa agttatttct 480 ggtgatccgt tcaattcaga tccagatgac caagaagtaa tgagtgcaga agtccgtgac 540 aaattgagga gcggacagca gcttcctata tctcgtacga aaatcaacat ggttgattta 600 ccgcttggtg ctactgagga cagggtgtgt ggcacaatcg acattgagaa agctcttact 660 gagggtgtga aggctttcga gcctggtctt cttgctaaag ctaacagagg aatactttac 720 gtcgatgagg ttaatctttt ggacgaccat ttagtagatg ttcttttgga ttctgcagca 780 tcgggatgga acactgttga aagagagggg atatcaatat cacaccctgc ccggtttatc 840 cttattggtt cgggtaatcc tgaagaagga gaacttaggc cacaacttct tgatcgattt 900 ggaatgcatg cccaagtggg gaccgtgaga gatgcagagc tgagagtgaa gatagttgag 960 gaaagagctc gttttgataa gaaccccaag gaattcaggg aatcatacaa ggcagagcaa 1020 gaaaagctcc agaaccaaat cgactcagct aggaacgctc tttctgctgt tacaatcgat 1080 catgatcttc gagttaaaat ctctaaggtc tgtgcagaac taaacgtcga tggattgaga 1140 ggtgatatag tcactaacag ggcagcaaga gcgttggctg cactaaaagg aagagataag 1200 gtaactccgg aagatatcgc cactgtcatt cccaactgct taagacacag gctgaggaag 1260 gatccgttgg aatctattga ctcgggtgta cttgttgttg agaaatttta tgaggttttc 1320 gcctaagcgt tttatagagt gagatactta tttttggctt tattttccat tcataaatca 1380 tctaaagatt tgacaattgt aacactagac ttttgcttaa ttttggcttt gtactgtgct 1440 taagaaatgg gttcagaatt acctgtagcc agttgtattt ggttatgact gctttatttc 1500 tgaaatgctt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaaaa aaggaattc 1579 6 4578 DNA Nicotiana tabacum misc_feature (1)..(4578) sequence for subunit chlH of plant magnesium chelatases 6 agccactact ctccacataa aactataaac ttagaacatt tcacttgaaa aaagagagga 60 aaaaagtgaa gcagaaatct tttctcaaaa cacaatctat aggaagttaa attcaacttc 120 cacacttcca agattcttgt ttcaagtttc gtttagtttt tttttcttgg ttttttttat 180 agttttctgt acaattttgt gtagaatcaa gaaacgaaag agttaaagtt tgaaactttt 240 ttacaagttt gaaacaatgg cttctttggt ttcttcacca tttacattgc caaattcaaa 300 agtagaacac ttgtcatcca tttctcaaaa gcattacttt cttcactcat ttcttcccaa 360 gaaaataaac cccacttact caaaatcacc aaagaaattc caatgtaatg ctattggcaa 420 tggtttattc actcaaacaa ctcaagaagt taggagaatt gtgcctgaaa atactcaggg 480 acttgctact gtgaaaatag tctatgttgt attggaagct cagtaccaat catcacttac 540 tgctgctgtt cagacactga acaagaatgg tcagtttgct tcttttgagg ttgtggggta 600 cttggttgag gagcttagag atgagaatac ttataaaatg ttttgtaaag atcttgagga 660 tgcaaatgtg tttattggtt cattgatttt tgtggaagaa ttggctttaa aggtaaaatc 720 tgcagtggag aaagaaaggg acagacttga tgcagttttg gtgtttccat caatgcctga 780 ggtgatgagg ttgaacaagt tgggatcttt tagtatgtca caattggggc aatcaaagag 840 tccatttttt gagcttttca agaagaagaa accttcttct gcaggttttt ctgatcagat 900 gttgaagctt gtgagaacat tgcctaaggt tttgaagtat ttaccaagtg ataaagctca 960 agatgctagg ttgtacatac taagtttgca gttttggcta ggaggttcac ctgataattt 1020 ggtgaatttc ttgaaaatga tttctggttc ttatgttcct gctcttaaag ggatgaaaat 1080 cgactactcg gatccggttt tgtacttgga taatggaatt tggcaccctt tggctccttg 1140 tatgtatgat gatgtgaagg agtatttgaa ttggtatgca acaaggagag atactaatga 1200 gaaactcaag agttcaaatg ctcctgttgt tgggctggtt ttgcaaagga gtcatattgt 1260 tacttgtgat gagagtcact atgtggctgt gatcatggaa ttggaggcaa agggggctaa 1320 agttatccca atttttgccg gtgggctaga cttttcgagg ccaattgaga gatatttcat 1380 tgatcctatt acaaagaagc cttttgtgaa ttcagtaata tcactttctg gttttgcact 1440 tgttggaggg ccagcaagac aagaccatcc aagggcaata gaggctttga tgaaacttga 1500 tgtgccttat attgtggcat tgcctttggt tttccaaaca acagaggaat ggttgaacag 1560 tactttgggg ctgcacccta ttcaggtggc tctacaagtt gctctccctg agctggatgg 1620 aggaatggag cccatcgtat tcgccggtcg cgatccaaga acagggaaat cacatgctct 1680 tcacaaaaga gtggagcagc tttgcaccag ggcaatcaaa tggggagagt taaagagaaa 1740 aacaaaggct gagaagaggt tggcaatcac tgtcttcagc tttcctccag acaaaggcaa 1800 tgtcggaact gctgcatact tgaatgtctt tgcctccata tactctgttc tcaaagatct 1860 caagaaagac ggctacaacg ttgaggggct gcctgagact tctgcacaac ttattgaaga 1920 agtaattcac gacaaagaag ctcagttcag cagcccaaat cttaacatag cttacaagat 1980 gaatgttaga gaataccaga agctaacccc ctatgctact gctcttgaag aaaactgggg 2040 gaaagcacct ggtaatttga actctgatgg agaaaacctc ttggtatatg gtaaacagta 2100 cggcaatgtc tttatcggtg ttcagcccac gtttggatac gagggtgacc cgatgagact 2160 tctgttctcc aaatcagcta gccctcacca tggttttgct gcatactatt cctttgtgga 2220 gaaaattttc aaagctgatg cagttctcca ctttggtact catggttctc ttgagttcat 2280 gccaggtaaa caggtgggaa tgagcgatgc ttctttccct gatagtctca ttggaaacat 2340 tcccaatgtc tattactatg cagcaaacaa cccatctgaa gcaactattg ccaaacgaag 2400 gagttatgcg aataccatta gctacttgac tcctccggct gagaatgctg gactctacaa 2460 gggactcaag cagctcagtg agctcatttc ctcataccaa tctctgaaag actcaggccg 2520 tggccaacag attgtgaact ctatcatcag tacagctaga cagtgtaatc ttgacaagga 2580 tgttgatctt ccagaagaag gggaggaaat ctcggccaaa gagcgtgacc ttgtggtagg 2640 aaaagtatac tctaagatta tggagatcga gtctcgtctt cttccgtgtg gacttcacat 2700 cattggtgaa cctccaaccg cgatggaagc agttgctact cttgtcaata ttgcgacatt 2760 ggaccgtcct gaagagggta tttctgccct tccatctata ttggctgcga cggttggaag 2820 aagcattgag gagatttaca gaggcaatga ccagggcatc ttacgagatg tggagctgct 2880 ccgtcaaatt actgaggcat cacgtggagc aatatcagca tttgttgaac gtacgacaaa 2940 caacaagggt caggttgtga atgtcaatga caagctaacc tcaatccttg gttttggtat 3000 aaatgaacca tggatccagt atttgtcaaa cacccaattt tacagagctg atagggacaa 3060 gctcagagtt ctattccagt tcttgggaga gtgtctgaag ctaattgtcg ctaacaacga 3120 ggtgggaagc ttgaaacagg ctctagaagg gaaatatgtt gaaccaggtc caggagggga 3180 tccgatcaga aacccgaaag ttttgcctac tgggaaaaac atccatgctt tggacccaca 3240 agctattccc acaatagcag cagtgcagag tgccaaaatt gttgttgaaa gattgttgga 3300 gaggcaaaag gccgacaacg ggggcaagta cccggagact gttgctctgg ttctttgggg 3360 aacagacaac atcaagacct atggagagtc attggcacag gttatgtgga tgattggtgt 3420 taggccagtt acagactcgt taggacgggt taaccgggtg gaacctgtta gccttgaaga 3480 gcttggaagg cctagagttg atgttgttgt caactgctct ggggtgttca gagatctctt 3540 catcaatcag atgaatctcc ttgaccgagc agtcaagatg gttgcagagc tcgacgagcc 3600 agaagaccaa aactacgtca ggaaacatgc actagaacaa gcaaaaacac tcggagttga 3660 tgttcgtgaa gctgctacaa ggatcttctc aaatgcttca ggatcttact cctccaacat 3720 taaccttgct gttgagaatt caacatggaa tgatgagaag caacttcaag acatgtactt 3780 gagccgaaag tcatttgcat ttgactgtga tgcccctggt gttggcatga ctgagaagag 3840 gaaagttttt gagatggctc ttagcacggc tgatgccaca ttccagaacc ttgactcatc 3900 tgaaatttca ttcacagacg tgagtcacta cttcgattca gacccaacca accttgtgca 3960 aaacctcagg aaagacggga agaagcctag tgcatacatt gctgacacca ctactgctaa 4020 tgctcaggta cgtacgttgt ctgagactgt gaggcttgac gcaaggacaa agttgttgaa 4080 ccccaagtgg tatgaaggca tgctatccac tggctacgag ggtgttcgtg agattgagaa 4140 acgattaact aacacagtgg ggtggagtgc aacttcaggc caagttgata attgggtgga 4200 tgaagaagcc aacacaacct tcattcaaga tcaggagatg ttgaacaggc tcatgaacac 4260 aaatccaaat tctttcagga agttgcttca gacattcttg gaagccaacg ggcgtggata 4320 ctgggaaact tctgcagaga acattgagaa actcaagcaa ttatactcag aagttgaaga 4380 caagattgag ggaatcgatc gataaatgta tagcaaaaag aatgatctct gattattgcc 4440 tgtttgttcc taactgtttc tgatgtgaat tcctttgaca gtccccagtg taattttgtt 4500 catttttggg gatgtcctac ttctatgaga aaatactgct tccatatatt caaatttgag 4560 cttgaaaaaa aaaaaaaa 4578 7 6 PRT Synechocystis PCC6803 PEPTIDE (1)..(6) peptide sequence derived from chlD gene of synechocystis PCC6803 7 Ala Lys Gly Ala Val Met 1 5 8 18 DNA Artificial PCR primer 8 gaygtngara arwcngtn 18 9 19 DNA Artificial PCR primer 9 atrttnccnc gnccrtcng 19 10 19 DNA Artificial PCR primer 10 gcnaarggng cngtnatgc 19 11 33 DNA Artificial Primer 11 tgacccgggg gtagtggaac ctgaaaaaca acc 33 12 33 DNA Artificial Primer 12 ggcgaattct caagattcct ttaatgcaga taa 33 13 30 DNA Artificial Primer 13 gcggtcgact caagattcct ttaatgcaga 30 14 33 DNA Artificial Primer 14 gctgatatcg gctattggca atggtttatt cac 33 15 30 DNA Artificial Primer 15 gcgtcgacat ttatcgatcg attccctcaa 30 16 21 DNA Artificial Primer 16 cagcccgggt ccactactag g 21 17 24 DNA Artificial Primer 17 caggtcgagg cacagtacaa agcc 24 

We claim:
 1. An isolated nucleic acid molecule that specifically hybridizes to SEQ ID NO: 1 or a fragment or a complement thereof, wherein the nucleic acid molecule encodes a plant magnesium (Mg) chelatase subunit CHLD.
 2. The nucleic acid molecule as claimed in claim 1 comprising SEQ ID NO: 1, which encodes a plant Mg chelatase subunit CHLD.
 3. The nucleic acid molecule as claimed in claim 1 which encodes a protein shown as SEQ ID NO:
 2. 4. An isolated nucleic acid molecule of at least 10 nucleotides in length which hybridizes specifically with the nucleic acid molecule as claimed in claim
 1. 5. An isolated nucleic acid molecule which encodes a plant peptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:
 7. 6. An isolated nucleic acid molecule that is an antisense or complementary sequence of the nucleic acid molecule as claimed in claim
 1. 7. A method of isolating or identifying a nucleic acid molecule encoding a protein with the function of a plant Mg chelatase subunit CHLD or a biologically active fragment thereof comprising isolating or identifying a nucleic acid molecule which specifically hybridizes to the isolated nucleic acid as claimed in claim 1 and which encodes a functional Mg chelatase subunit CHLD.
 8. A method of generating a protein with the function of a plant Mg chelatase subunit CHLD comprising: i. transforming a host cell with a nucleic acid molecule according to claim 1; ii. growing the host cell in a suitable medium; iii. expressing the nucleic acid molecule encoding a plant Mg chelatase subunit CHLD or a biologically active fragment thereof; and purifying the plant Mg chelatase subunit CHLD or biologically active fragment thereof in a suitable manner from the culture medium or from the host cell.
 9. A method of generating a plant Mg chelatase comprising: i. transforming a host cell with a nucleic acid molecule according to claim 1 and at least one nucleic acid molecule encoding a further plant Mg chelatase subunit; ii. growing the host cell in a suitable medium; iii. expressing the nucleic acid molecules encoding the plant Mg chelatase subunits; and iv. purifying the plant Mg chelatase or biologically active fragment thereof in a suitable manner from the culture medium or from the host cell.
 10. A purified recombinant protein having the function of a plant Mg chelatase subunit CHLD.
 11. The purified protein as claimed in claim 10, which comprises SEQ ID NO:
 2. 12. A method for determining Mg chelatase activity comprising measuring the activity of the purified protein as claimed in claim 10 in a suitable expression system.
 13. A non-naturally occurring chimeric gene comprising a promoter functionally fused to the nucleic acid molecule as claimed in claim
 1. 14. A vector comprising a nucleic acid molecule as claimed in claim
 1. 15. A recombinant host cell which expresses a nucleic acid molecule as claimed in claim
 1. 16. A transgenic plant, transgenic plant cells, transgenic plant organs, transgenic plant seeds, or transgenic propagation material comprising a nucleic acid molecule as claimed in claim
 1. 17. The recombinant host cell according to claim 15, which is stably transformed with the vector of claim
 14. 